Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 13 de 13
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
2.
Nature ; 549(7672): 351-356, 2017 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-28902842

RESUMO

Type 2 innate lymphoid cells (ILC2s) both contribute to mucosal homeostasis and initiate pathologic inflammation in allergic asthma. However, the signals that direct ILC2s to promote homeostasis versus inflammation are unclear. To identify such molecular cues, we profiled mouse lung-resident ILCs using single-cell RNA sequencing at steady state and after in vivo stimulation with the alarmin cytokines IL-25 and IL-33. ILC2s were transcriptionally heterogeneous after activation, with subpopulations distinguished by expression of proliferative, homeostatic and effector genes. The neuropeptide receptor Nmur1 was preferentially expressed by ILC2s at steady state and after IL-25 stimulation. Neuromedin U (NMU), the ligand of NMUR1, activated ILC2s in vitro, and in vivo co-administration of NMU with IL-25 strongly amplified allergic inflammation. Loss of NMU-NMUR1 signalling reduced ILC2 frequency and effector function, and altered transcriptional programs following allergen challenge in vivo. Thus, NMUR1 signalling promotes inflammatory ILC2 responses, highlighting the importance of neuro-immune crosstalk in allergic inflammation at mucosal surfaces.


Assuntos
Hipersensibilidade/imunologia , Hipersensibilidade/patologia , Inflamação/imunologia , Inflamação/patologia , Pulmão/patologia , Linfócitos/imunologia , Neuropeptídeos/metabolismo , Animais , Feminino , Regulação da Expressão Gênica , Imunidade Inata/imunologia , Interleucina-17/imunologia , Interleucina-33/imunologia , Ligantes , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Neurotransmissores/biossíntese , Receptores de Neurotransmissores/genética , Receptores de Neurotransmissores/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Transdução de Sinais , Transcrição Gênica
3.
Science ; 353(6302): 925-8, 2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27471252

RESUMO

Single-cell RNA sequencing (RNA-Seq) provides rich information about cell types and states. However, it is difficult to capture rare dynamic processes, such as adult neurogenesis, because isolation of rare neurons from adult tissue is challenging and markers for each phase are limited. Here, we develop Div-Seq, which combines scalable single-nucleus RNA-Seq (sNuc-Seq) with pulse labeling of proliferating cells by 5-ethynyl-2'-deoxyuridine (EdU) to profile individual dividing cells. sNuc-Seq and Div-Seq can sensitively identify closely related hippocampal cell types and track transcriptional dynamics of newborn neurons within the adult hippocampal neurogenic niche, respectively. We also apply Div-Seq to identify and profile rare newborn neurons in the adult spinal cord, a noncanonical neurogenic region. sNuc-Seq and Div-Seq open the way for unbiased analysis of diverse complex tissues.


Assuntos
Núcleo Celular/metabolismo , Neurogênese/genética , Neurônios/citologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma , Animais , Divisão Celular/genética , Desoxiuridina/análogos & derivados , Desoxiuridina/análise , Hipocampo/citologia , Hipocampo/metabolismo , Marcação por Isótopo , Camundongos , Neurônios/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , Transcrição Gênica
4.
Science ; 352(6282): 189-96, 2016 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-27124452

RESUMO

To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.


Assuntos
Melanoma/genética , Melanoma/secundário , Neoplasias Cutâneas/patologia , Microambiente Tumoral , Sequência de Bases , Comunicação Celular , Ciclo Celular , Resistencia a Medicamentos Antineoplásicos/genética , Células Endoteliais/patologia , Genômica , Humanos , Imunoterapia , Ativação Linfocitária , Melanoma/terapia , Fator de Transcrição Associado à Microftalmia/metabolismo , Metástase Neoplásica , RNA/genética , Análise de Sequência de RNA , Análise de Célula Única , Células Estromais/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , Transcriptoma
5.
Cell ; 162(6): 1309-21, 2015 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-26343579

RESUMO

Encounters between immune cells and invading bacteria ultimately determine the course of infection. These interactions are usually measured in populations of cells, masking cell-to-cell variation that may be important for infection outcome. To characterize the gene expression variation that underlies distinct infection outcomes and monitor infection phenotypes, we developed an experimental system that combines single-cell RNA-seq with fluorescent markers. Probing the responses of individual macrophages to invading Salmonella, we find that variation between individual infected host cells is determined by the heterogeneous activity of bacterial factors in individual infecting bacteria. We illustrate how variable PhoPQ activity in the population of invading bacteria drives variable host type I IFN responses by modifying LPS in a subset of bacteria. This work demonstrates a causative link between host and bacterial variability, with cell-to-cell variation between different bacteria being sufficient to drive radically different host immune responses. This co-variation has implications for host-pathogen dynamics in vivo.


Assuntos
Interações Hospedeiro-Patógeno , Macrófagos/imunologia , Salmonella typhimurium/fisiologia , Animais , Interferon Tipo I/imunologia , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Organismos Livres de Patógenos Específicos
6.
Cell ; 161(5): 1202-1214, 2015 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-26000488

RESUMO

Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell's RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts' cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution. VIDEO ABSTRACT.


Assuntos
Perfilação da Expressão Gênica/métodos , Estudo de Associação Genômica Ampla , Técnicas Analíticas Microfluídicas , Retina/citologia , Análise de Célula Única , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Análise de Sequência de RNA
7.
Nature ; 516(7529): 56-61, 2014 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-25471879

RESUMO

Pluripotent stem cells (PSCs) are capable of dynamic interconversion between distinct substates; however, the regulatory circuits specifying these states and enabling transitions between them are not well understood. Here we set out to characterize transcriptional heterogeneity in mouse PSCs by single-cell expression profiling under different chemical and genetic perturbations. Signalling factors and developmental regulators show highly variable expression, with expression states for some variable genes heritable through multiple cell divisions. Expression variability and population heterogeneity can be influenced by perturbation of signalling pathways and chromatin regulators. Notably, either removal of mature microRNAs or pharmacological blockage of signalling pathways drives PSCs into a low-noise ground state characterized by a reconfigured pluripotency network, enhanced self-renewal and a distinct chromatin state, an effect mediated by opposing microRNA families acting on the Myc/Lin28/let-7 axis. These data provide insight into the nature of transcriptional heterogeneity in PSCs.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes/fisiologia , Animais , Morte Celular , Divisão Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Perfilação da Expressão Gênica , Camundongos , MicroRNAs/metabolismo , Células-Tronco Pluripotentes/citologia , Transdução de Sinais
8.
Curr Protoc Mol Biol ; 107: 4.22.1-4.22.17, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24984854

RESUMO

For the past several decades, due to technical limitations, the field of transcriptomics has focused on population-level measurements that can mask significant differences between individual cells. With the advent of single-cell RNA-Seq, it is now possible to profile the responses of individual cells at unprecedented depth and thereby uncover, transcriptome-wide, the heterogeneity that exists within these populations. This unit describes a method that merges several important technologies to produce, in high-throughput, single-cell RNA-Seq libraries. Complementary DNA (cDNA) is made from full-length mRNA transcripts using a reverse transcriptase that has terminal transferase activity. This, when combined with a second "template-switch" primer, allows for cDNAs to be constructed that have two universal priming sequences. Following preamplification from these common sequences, Nextera XT is used to prepare a pool of 96 uniquely indexed samples ready for Illumina sequencing.


Assuntos
Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Mensageiro , Análise de Sequência de RNA/métodos , Animais , DNA Complementar/química , DNA Complementar/genética , Humanos , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação
9.
Nature ; 510(7505): 363-9, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24919153

RESUMO

High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis and function of gene expression variation between seemingly identical cells. Here we sequence single-cell RNA-seq libraries prepared from over 1,700 primary mouse bone-marrow-derived dendritic cells spanning several experimental conditions. We find substantial variation between identically stimulated dendritic cells, in both the fraction of cells detectably expressing a given messenger RNA and the transcript's level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a 'core' module of antiviral genes is expressed very early by a few 'precocious' cells in response to uniform stimulation with a pathogenic component, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analysing dendritic cells from knockout mice, and modulating secretion and extracellular signalling, we show that this response is coordinated by interferon-mediated paracrine signalling from these precocious cells. Notably, preventing cell-to-cell communication also substantially reduces variability between cells in the expression of an early-induced 'peaked' inflammatory module, suggesting that paracrine signalling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations can use to establish complex dynamic responses.


Assuntos
Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade/genética , Comunicação Parácrina , Animais , Antígenos Virais/farmacologia , Sequência de Bases , Comunicação Celular , Células Dendríticas/efeitos dos fármacos , Perfilação da Expressão Gênica , Interferon beta/genética , Camundongos , Técnicas Analíticas Microfluídicas , Análise de Componente Principal , RNA Mensageiro/química , RNA Mensageiro/genética , Análise de Célula Única
10.
Science ; 344(6190): 1396-401, 2014 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-24925914

RESUMO

Human cancers are complex ecosystems composed of cells with distinct phenotypes, genotypes, and epigenetic states, but current models do not adequately reflect tumor composition in patients. We used single-cell RNA sequencing (RNA-seq) to profile 430 cells from five primary glioblastomas, which we found to be inherently variable in their expression of diverse transcriptional programs related to oncogenic signaling, proliferation, complement/immune response, and hypoxia. We also observed a continuum of stemness-related expression states that enabled us to identify putative regulators of stemness in vivo. Finally, we show that established glioblastoma subtype classifiers are variably expressed across individual cells within a tumor and demonstrate the potential prognostic implications of such intratumoral heterogeneity. Thus, we reveal previously unappreciated heterogeneity in diverse regulatory programs central to glioblastoma biology, prognosis, and therapy.


Assuntos
Neoplasias Encefálicas/genética , Variação Genética , Glioblastoma/genética , RNA Mensageiro/genética , Neoplasias Encefálicas/classificação , Neoplasias Encefálicas/tratamento farmacológico , Perfilação da Expressão Gênica , Glioblastoma/classificação , Glioblastoma/tratamento farmacológico , Humanos , Prognóstico , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos
11.
Nat Biotechnol ; 32(5): 479-84, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24752078

RESUMO

Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two patients with prostate cancer, including one for whom we sequenced CTCs, a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were present in matched tissue. Moreover, we identified 10 early trunk and 56 metastatic trunk mutations in the non-CTC tumor samples and found 90% and 73% of these mutations, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic.


Assuntos
Exoma/genética , Células Neoplásicas Circulantes , Neoplasias da Próstata/genética , Humanos , Masculino , Mutação/genética
12.
Nature ; 498(7453): 236-40, 2013 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-23685454

RESUMO

Recent molecular studies have shown that, even when derived from a seemingly homogenous population, individual cells can exhibit substantial differences in gene expression, protein levels and phenotypic output, with important functional consequences. Existing studies of cellular heterogeneity, however, have typically measured only a few pre-selected RNAs or proteins simultaneously, because genomic profiling methods could not be applied to single cells until very recently. Here we use single-cell RNA sequencing to investigate heterogeneity in the response of mouse bone-marrow-derived dendritic cells (BMDCs) to lipopolysaccharide. We find extensive, and previously unobserved, bimodal variation in messenger RNA abundance and splicing patterns, which we validate by RNA-fluorescence in situ hybridization for select transcripts. In particular, hundreds of key immune genes are bimodally expressed across cells, surprisingly even for genes that are very highly expressed at the population average. Moreover, splicing patterns demonstrate previously unobserved levels of heterogeneity between cells. Some of the observed bimodality can be attributed to closely related, yet distinct, known maturity states of BMDCs; other portions reflect differences in the usage of key regulatory circuits. For example, we identify a module of 137 highly variable, yet co-regulated, antiviral response genes. Using cells from knockout mice, we show that variability in this module may be propagated through an interferon feedback circuit, involving the transcriptional regulators Stat2 and Irf7. Our study demonstrates the power and promise of single-cell genomics in uncovering functional diversity between cells and in deciphering cell states and circuits.


Assuntos
Células Dendríticas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Splicing de RNA/imunologia , Análise de Célula Única , Transcriptoma/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Hibridização in Situ Fluorescente , Fator Regulador 7 de Interferon , Interferons/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Reprodutibilidade dos Testes , Fator de Transcrição STAT2 , Análise de Sequência de RNA , Vírus/imunologia
13.
Nature ; 496(7446): 461-8, 2013 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-23467089

RESUMO

Despite their importance, the molecular circuits that control the differentiation of naive T cells remain largely unknown. Recent studies that reconstructed regulatory networks in mammalian cells have focused on short-term responses and relied on perturbation-based approaches that cannot be readily applied to primary T cells. Here we combine transcriptional profiling at high temporal resolution, novel computational algorithms, and innovative nanowire-based perturbation tools to systematically derive and experimentally validate a model of the dynamic regulatory network that controls the differentiation of mouse TH17 cells, a proinflammatory T-cell subset that has been implicated in the pathogenesis of multiple autoimmune diseases. The TH17 transcriptional network consists of two self-reinforcing, but mutually antagonistic, modules, with 12 novel regulators, the coupled action of which may be essential for maintaining the balance between TH17 and other CD4(+) T-cell subsets. Our study identifies and validates 39 regulatory factors, embeds them within a comprehensive temporal network and reveals its organizational principles; it also highlights novel drug targets for controlling TH17 cell differentiation.


Assuntos
Diferenciação Celular/genética , Redes Reguladoras de Genes/genética , Células Th17/citologia , Células Th17/metabolismo , Animais , Células Cultivadas , DNA/genética , DNA/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Técnicas de Silenciamento de Genes , Genoma/genética , Interferon gama/biossíntese , Interleucina-2/genética , Camundongos , Camundongos Endogâmicos C57BL , Nanofios , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Silício , Células Th17/imunologia , Fatores de Tempo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Receptor fas/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...